Antimicrobial Efficacy of Acacia nilotica Against Microorganisms of Oral Cavity- An In Vitro Study

Introduction

Oral health is the window to an individual’s overall health. Good oral health contributes positively to one’s physical, mental and social well-being.¹ The major bulk of the world population is afflicted by periodontal diseases and dental caries.² History of Indian medicine goes back to the age of Vedas and Puranas. India has witnessed the Ayurvedic period of Sushrutha, Charaka and Vaghbatta. Honey, mango leaf, neem, triphala, turmeric, Amaranthus spinosus, Azadirachta indica, and pomegranate etc. have antimicrobial activity against dental caries.³

Many oral and dental infections such as periodontal diseases, gingivitis, pericoronitis, endodontitis, periimplantitis, and post extraction infections are due to the colonization of microbial pathogens.⁴

Acacia belongs to pantropical and subtropical genus with the sub-family Mimosoideae of the family Fabaceae. It is native of Africa, the Middle East and the Indian subcontinent. Acacia nilotica is a thorny, medium sized, tree found in the India regions with less rain. The bark is used mainly for toothache, cold, bronchitis, diarrhoea, bleeding piles and leukoderma. The tender twigs are used as tooth brushes.⁵

Acacia nilotica was chosen for this study because of its wide range of therapeutic uses such as antimicrobial, antiplasmodial, antioxidant properties. The phytochemical constituents of this plant were investigated with an aim to identify the secondary metabolites which reflect the antimicrobial effects of A. nilotica plant.

Different extracts from traditional plants have been tested to identify the source of therapeutic effects on oral cavity. Literature on the antibacterial potential of pod, leaves and bark (extracts) of Acacia nilotica in the oral cavity has been reported. Hence this study was conducted to compare the antimicrobial efficacy of flower and bark extracts of Acacia nilotica against microorganisms of oral cavity.

Materials and Methods

Study design and study setting

An in vitro experimental study was conducted from February to July 2022 at a research lab in Bangalore.

Sample size

A sample size of 54 (18 per group, three per concentration) would yield 90% power to detect significant differences, with assuming the effect size of 0.5 and significance level at 0.05.

(Group1- flower, Group 2- bark, Group 3- chlorhexidine)

Plant material

The flower and bark were acquired from the local areas and were identified and authenticated by the Department of Dravyaguna, SDM Institute of Ayurveda and Hospital, Bangalore.

The flowers were thoroughly washed with distilled water at room temperature, and a hot air oven was used to dry at 24 to 72 °C. The dried flower was uniformly ground using an electric grinder.¹

Preparation of extract

10g of powdered sample was filled into a thimble and subjected to Soxhlet extraction6 using 150 mL methanol as solvent. A rotary evaporator was employed to concentrate the extract for further analysis.

Test microorganism

Test microorganisms Streptococcus mutans (MTCC 890), Lactobacillus acidophilus (MTCC 10307) used in this study were obtained from a private research lab in Bangalore.

Antimicrobial testing

The antimicrobial activity of plant extract was determined by using Agar well diffusion⁷ method. A 6 mm diameter wells were punched on specific agar media. About 100 μL of pre-cultured test organisms-Streptococcus mutans and Lactobacillus acidophilus, were spread onto the agar plates. Various concentrations (5 mg, 10 mg, 15 mg, 20 mg, 30 mg, 40 mg) of samples were loaded into the wells. Duplicated bacterial plates were incubated at 37⁰C for 24 hours, and the inhibition zones were measured and tabulated.

Minimum Inhibitory Concentration (MIC)

The MIC of the herbal extract against S. mutans and L. acidophilus was determined by micro dilution assay using a separate microplate for each bacterial species and incubated for 24 h at 37oC. After incubation, the optical density of each well was assessed using a spectrophotometer (Labman, India) at 600 nm.4 MIC of the extract that repressed the turbidity was determined.⁸

Minimum Bactericidal Concentration (MBC)

The MBC⁹ was determined by sub culturing the contents of the wells that displayed no apparent growth on a sterile agar plate. 100 µL of the bacterial solution which was considered as the MIC and higher concentrations were grown on tryptone soya agar plates. The plates were incubated for 24 hours at 37oC. Following incubation each plate was observed for growth at the end of incubation period both with the naked eye, by counting Colony Forming Units (CFUs)¹⁰ which were calculated on a grid. The MBC value was concluded as the lowest concentration which showed no apparent growth on the agar plate.⁶ Data entry and statistical analysis SPSS (Statistical Package For Social Sciences) version 20. (IBM SPSS statistics [IBM corp. released 2011] was used to perform the statistical analysis.

Results

Phytochemical analysis¹¹ results of flower extract and Retention Factor (RF) value of A. nilotica are shown in Table 1. They reveal the presence of certain secondary metabolites¹² such as alkaloids, flavonoid, glycosides, tannins, terpenoids and steroids, while saponin was absent in it.

The inhibition zone of flower extract of A. nilotica is illustrated in Table 2. The zones of inhibition at 5 mg/ mL, 10 mg/mL, 15 mg/mL, 20 mg/mL, 30 mg/mL, 40 mg/mL were sized 6 mm, 8 mm, 10 mm, 12 mm, 15 mm and 21 mm, respectively. Chlorhexidine 0.2% (Group 3) produced least inhibition zone (13.85 mm) against S. mutans (Figure 1 & 2).

The results of normality test presented in Table 3 shows that the antimicrobial efficacy of flower extract of Acacia nilotica against S. mutans and L. acidophilus was statistically significant.

The results revealed that the MIC OD at 600 nm showed maximum inhibition at 40 mg/mL against S. mutans and L. acidophilus which was about 78% and 91%, respectively. MBC of flower extract at 40 mg/mL against S. mutans and L. acidophilus produced 1 and 2 CFU, respectively.

The results of comparison of the microorganisms between the groups are presented in Table 4. It was found that the comparison between the groups against L. acidophilus was statistically significant than the comparison between the groups against S. mutans.

Discussion

This study was conducted to examine the antibacterial efficacy of flower extract and bark extract of Acacia nilotica against S. mutans and L. acidophilus. The plants contents such as phenolic compounds, tannins,saponins, essential oils and flavonoids have antimicrobial potency.¹³

It has been suggested that the mechanism of the antimicrobial effects involves inhibition in different cellular processes, like a raise in plasma membrane permeability and ultimately, ion seepage from the cells.¹⁴ Attahiru et al. ⁷ (2021) showed that the preliminary qualitative phytochemical screening of crude methanol extracts of A. nilotica leaves gave positive test for alkaloids, glycosides, cardiac glycoside, steroids, saponins, tannins, anthraquinones, flavonoids, terpenoids and phenol. Our study showed that the flower also gave positive results for alkaloids, tannins, glycosides, steroids, saponins.

Arshad et al, ⁶ revealed that the methanol extract of A. nilotica showed significantly higher zones of inhibition (18.00±1.00 mm, 20.00±1.15 mm and 16.67±0.67 mm) and MIC (0.3125, 0.3125 and 0.15625 mg/mL), followed by acetone and aqueous extracts against S. mutans, S. mitis and P. intermedia, respectively. The current study found that the flower extract of A. nilotica demonstrated a significantly higher antimicrobial efficacy in terms of zone of inhibition and MIC when compared to bark extract against S. mutans and L. acidophilus.

Deshpande and Kadam¹ reported Acacia nilotica, ethanolic bark extract to inhibit the growth of S. mutans (31±0.7 mm). In the present study, we found that methanolic flower extract of Acacia nilotica inhibited the growth of S. mutans and L. acidophilus.

Chandra Shekar et al.,² concluded that combination of leaves extracts of Acacia nilotica and P. guajava produced highest mean diameter of zone of inhibition (21.08±2.11 mm) against Streptococcus mutans while chlorhexidine produced the least zone of inhibition against S. mutans (14.50±2.07 mm). The Acacia nilotica and P. guajava combination produced the maximum antimicrobial efficacy against S. sanguis (19.67 ± 1.03) and S. salivarius (20.33±1.86 mm). Our findings showed that Acacia nilotica alone is capable of inhibiting bacterial growth which produced highest inhibition zone (21 mm) at 40 mg/mL against S. mutans. Our study revealed that the antimicrobial efficacy of flower extract of Acacia nilotica produced highest zone of inhibition (21 mm) against S. mutans when compared with 0.2% chlorhexidine (14.5 mm).

Pote and Hirapure⁴ stated that all the three extracts (pod, leaves, bark) of Acacia nilotica showed highest antimicrobial potential against Streptococcus sanguinis followed by Streptococcus salivarius and lowest antimicrobial potential was observed against Lactobacillus acidophilus. The highest antimicrobial efficacy was observed with the pod extract followed by leaf extract and bark extract against all test microorganisms. This study found that flower extract of Acacia nilotica demonstrated highest antimicrobial efficacy when compared to other extracts against S. mutans and L. acidophilus.

Chandra Shekar et al.,¹⁵ demonstrated MIC of A. nilotica, M. koenigii, Eucalyptus hybrid, and P. guajava combinations against S. mutans, L. acidophilus (dental caries bacteria), S. sanguis, S. salivarius, F. nucleatum, and P. gingivalis to be 0.25, 0.05, 0.05, 0.1, 0.25, and 0.25%, respectively. Our findings revealed that Acacia nilotica alone showed maximum inhibition at MIC of 40 mg/mL against S. mutans and L. acidophilus of about 78% and 91%, respectively.

Abduljawad A¹⁶ (2020) showed that Acacia has many therapeutic effects including antimicrobial, antiparasitic, antidiabetic, antihyperlipidemic, anticancer, antimutagenic, antipyretic, anti-inflammatory, antinociceptive, antiulcer, antihypertensive, antispasmodic and antidiarrheal, and antioxidant activities. Therefore, Acacia extracts can be a natural, inexpensive replacement to pharmaceuticals and therapeutics.

Pai M B H et al ¹⁷ showed that Punica granatum exhibited the highest inhibition of C albicans with a mean zone of inhibition of 22 mm and compared with acacia and other plants whereas in the present study Acacia nilotica itself showed 21mm zone of inhibition.

The present study with its strength being the first of its kind was done using flower extract to determine the antimicrobial efficacy against organisms of oral cavity.

Recommendations

In the present study, we included only S. mutans and L. acidophilus as organisms of oral cavity. In the future studies, more microorganism from the oral cavity can be considered.

Conclusion

Acacia nilotica was selected because of its reputation in medicine as antimicrobial agent and its use in many diseases. We here reported the antimicrobial efficacy of flower extract of Acacia nilotica against dental pathogens - S. mutans and L. acidophilus.

Phytochemical analysis of flower extract and RF value of A. nilotica were documented. It reveals the presence of certain secondary metabolites such as alkaloids, flavonoids, glycosides, tannins, terpenoids and steroids.

The study found that the extracts were active in inhibiting the growth of S. mutans, L. acidophilus. The efficacy of A. nilotica against S. mutans and L. acidophilus was significantly higher showing the zones of inhibition of 21 mm and 19 mm, respectively at 40 mg/mL. These have the potential to be used as anticaries and antiplaque agents.

MIC OD at 600 nm showed maximum inhibition of 78% and 91% at 40 mg/mL against S. mutans (0.437) and L. acidophilus (0.1545), respectively. MBC of flower extract at 40 mg/mL against S. mutans and L. acidophilus produced 1 and 2 CFU, respectively.

The current study evaluated the antimicrobial potential of flower extract and compared with the extracts of other parts of Acacia nilotica against the dental pathogens- S. mutans, L. acidophilus.

The presented study signifies the efficacy of flower of Acacia nilotica as the source of therapeutic agents, which may provide a route in the ongoing search for antibacterial agents from plants. This study suggests that Acacia nilotica might be used in the preparation of mouth wash or tooth paste to prevent oral diseases.

Funding

The authors are also thankful to the Institution, for providing this research grant.

Conflict of interest

None

Acknowledgement

The authors are grateful to Department of Dravyaguna, SDM Institute of Ayurveda and Hospital Bangalore for support of this work. We sincerely thank the management of Dextrose Lab Pvt Ltd, Bangalore for their kind permission and co operation in completing this research project.